Polyethylene Glycol-Mediated Directional Conjugation of Biological Molecules for Enhanced Immunoassays at the Point-of-Care

Rapid and reliable point-of-care (POC) diagnostic tests can have a significant impact on global health. One of the most common approaches for developing POC systems is the use of target-specific biomolecules. However, the conjugation of biomolecules can result in decreased activity, which may compromise the analytical performance and accuracy of the developed systems. To overcome this challenge, we present a polymer-based cross-linking protocol for controlled and directed conjugation of biological molecules. Our protocol utilizes a bifunctional thiol-polyethylene glycol (PEG)-hydrazide polymer to enable site-directed conjugation of IgG antibodies to the surface of screen-printed metal electrodes. The metal surface of the electrodes is first modified with thiolated PEG molecules, leaving the hydrazide groups available to react with the aldehyde group in the Fc fragments of the oxidized IgG antibodies. Using anti-Klebsiella pneumoniae carbapenemase-2 (KPC-2) antibody as a model antibody used for antimicrobial resistance (AMR) testing, our results demonstrate a ~10-fold increase in antibody coupling compared with the standard N-hydroxysuccinimide (NHS)-based conjugation chemistry and effective capture (>94%) of the target KPC-2 enzyme antigen on the surface of modified electrodes. This straightforward and easy-to-perform strategy of site-directed antibody conjugation can be engineered for coupling other protein- and non-protein-based biological molecules commonly used in POC testing and development, thus enhancing the potential for improved diagnostic accuracy and performance.


Introduction
The demand for point-of-care (POC) medicine is rapidly increasing in our modernized and globalized world. POC diagnostics have revolutionized healthcare by providing rapid and accurate disease diagnosis at the patient's bedside or in a clinical setting [1]. Immunoassays are widely used in POC diagnostics for detecting analytes such as proteins, nucleic acids, and small molecules [2]. However, the sensitivity and specificity of immunoassays

Results and Discussion
The successful development of effective POC diagnostics relies on the use of optimized conjugation methods that enable the selective and controlled conjugation of antibodies to the assay platform [32,33]. This can significantly improve the sensitivity and specificity of immunoassays, enabling accurate and reliable disease diagnosis at the POC [1,34]. Over the past decade, there has been a significant advancement in engineering PEG chemistry [19,35]. A majority of current applications for PEG include therapeutic and diagnostic [16,17]. PEGylation of antibodies, in particular, has been known to increase the half-life and reduce the non-specific binding of antibodies, which hence leads to the economical production and usage of immunoassays [36,37]. Here, we describe a protocol for the site-directed conjugation of a bifunctional 20 kDa PEG linker to the Fc region of polyclonal anti-KPC-2 antibody to assist binding to gold screen-printed electrodes (Au-SPEs), one of the most common platforms used for electrochemical-based immunoassays at the POC [38][39][40]. This approach resulted in a packed conjugation of antibody molecules on the surface of Au-SPE, which is more favorable as there are more functional moieties to bind the antigen molecule. This also achieved enhanced capture of the KPC-2 antibody, supporting the potential for sensitive detection of antimicrobial resistance (AMR) caused by carbapenemases.
To develop screen-printed electrodes for the detection of KPC-2 enzyme at POC, we designed a site-directed conjugation chemistry that relies on a heterobifunctional PEG polymer that links oxidized polyclonal anti-KPC-2 antibodies to the surface of Au-SPEs [41,42] ( Figure 1a). PEGylation of the Au-SPE was conducted using thiolated-PEG-hydrazide (SH-PEG-hydrazide), which forms a non-covalent gold-thiol bond, leaving the PEG-hydrazide arm free to react [43]. The anti-KPC-2 polyclonal antibody was oxidized using sodium metaperiodate to convert the carbohydrate group in glycoproteins to reactive aldehyde groups. The free hydrazide group on the surface of Au-SPE reacted with the aldehyde group on the antibody Fc region for its directional conjugation (Figure 1a). improved conjugation and target-capture efficiency compared to conventional conjugation methods. In our approach, a heterobifunctional linker of thiol-PEG-hydrazide (SH-PEG-hydrazide) was used to modify the carbohydrate residues of the Fc region of the polyclonal anti-KPC-2 IgG antibody. The antibody modified with PEG-thiol is then allowed to react with the surface of Au electrodes via the well-known thiol-metal bonding chemistry. Our approach represents a significant advancement in the development of conjugation chemistry for POC diagnostics, offering improved performance and accuracy for rapid and reliable disease diagnosis.

Results and Discussion
The successful development of effective POC diagnostics relies on the use of optimized conjugation methods that enable the selective and controlled conjugation of antibodies to the assay platform [32,33]. This can significantly improve the sensitivity and specificity of immunoassays, enabling accurate and reliable disease diagnosis at the POC [1,34]. Over the past decade, there has been a significant advancement in engineering PEG chemistry [19,35]. A majority of current applications for PEG include therapeutic and diagnostic [16,17]. PEGylation of antibodies, in particular, has been known to increase the half-life and reduce the non-specific binding of antibodies, which hence leads to the economical production and usage of immunoassays [36,37]. Here, we describe a protocol for the site-directed conjugation of a bifunctional 20 kDa PEG linker to the Fc region of polyclonal anti-KPC-2 antibody to assist binding to gold screen-printed electrodes (Au-SPEs), one of the most common platforms used for electrochemical-based immunoassays at the POC [38][39][40]. This approach resulted in a packed conjugation of antibody molecules on the surface of Au-SPE, which is more favorable as there are more functional moieties to bind the antigen molecule. This also achieved enhanced capture of the KPC-2 antibody, supporting the potential for sensitive detection of antimicrobial resistance (AMR) caused by carbapenemases.
To develop screen-printed electrodes for the detection of KPC-2 enzyme at POC, we designed a site-directed conjugation chemistry that relies on a heterobifunctional PEG polymer that links oxidized polyclonal anti-KPC-2 antibodies to the surface of Au-SPEs [41,42] (Figure 1a). PEGylation of the Au-SPE was conducted using thiolated-PEG-hydrazide (SH-PEG-hydrazide), which forms a non-covalent gold-thiol bond, leaving the PEGhydrazide arm free to react [43]. The anti-KPC-2 polyclonal antibody was oxidized using sodium metaperiodate to convert the carbohydrate group in glycoproteins to reactive aldehyde groups. The free hydrazide group on the surface of Au-SPE reacted with the aldehyde group on the antibody Fc region for its directional conjugation (Figure 1a).  To evaluate the efficiency of the developed PEG-based site-directed conjugation approach, we tested the presence of antibodies on the surface of Au-SPEs using multiple techniques, including colorimetry, fluorometry, Ultraviolet-visible (UV-Vis), Fourier-transform infrared (FTIR) spectroscopy, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis [44]. The digital images captured for the unmodified (i.e., no PEG and/or antibody) and PEG/antibody-modified Au-SPEs showed a change in the color of the modified electrode surface (Figure 1b). In addition, the Au-SPEs were digested using tris (2-carboxyethyl) phosphine (TCEP), and the UV-Vis spectrophotometry technique was used to confirm the presence of antibodies in the eluted solution [45][46][47]. The characteristic absorbance peak for PEG was observed near 220 nm, and the absorbance peak for the antibody was observed near 260 nm, compared with unmodified control Au-SPEs ( Figure 2c). To further characterize the anti-KPC-2 IgG antibody-modified Au-SPEs, a horseradish peroxidase (HRP)-based colorimetry technique was used. The unmodified and anti-KPC-2 IgG-modified electrodes were incubated with HRP-conjugated anti-IgG for 15 min, and then they were gently washed three times, and then 10 µL of 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate was added to the Au-SPEs ( Figure 2a). The complex was incubated with TMB for 10 min, and a strong blue color was seen in the antibody-modified SPE. This suggests that the HRP-bound anti-IgG was able to successfully bind the anti-KPC-2 antibody and was able to reduce the TMB to produce a blue color. The TMB added to the unmodified SPE did not produce any color change, indicating the absence of anti-KPC-2 antibodies. Further, the reduced TMB substrate was collected, and the absorbance of the formed complex was measured at 652 nm. The sharp rise in the absorbance at 652 nm confirmed the presence of anti-KPC-2 antibodies on the surface of Au-SPE and its labeling with HRP. The KPC-2 antibody-modified Au-SPEs were also incubated with protein-G coupled with fluorescein isothiocyanate (FITC) for 15 min (Figure 3a). Protein G tends to bind to most IgG molecules at near physiological pH at room temperature (25 • C). The bound antibody complex was then digested using TCEP from both unmodified and modified SPEs to release the IgG-protein G-FITC complex from the surface of Au-SPE, and the fluorescence signal was measured for the released complex at 528 nm. The coupled FITC to protein G was able to produce a fluorescent signal at 528 nm, which was~68.2% higher than the control sample of unmodified electrodes, indicating a successful conjugation of antibody to the surface of Au-SPE.
PEG-hydrazide followed by coupling with an antibody. (b) Digital images of unmodified (top) and modified Au-SPE (bottom), showing a change in color of the surface post-modification. (c) UV-Vis spectrum of control Au-SPE (without any treatment; black line), PEGylated Au-SPE (red line), and antibody-coupled Au-SPE (blue line). The surface of Au-SPEs was digested with TCEP, and the eluted solution was tested using a NanoDrop spectrophotometer (Thermofisher Scientific Inc, Waltham, USA) to measure the absorbance.
To evaluate the efficiency of the developed PEG-based site-directed conjugation approach, we tested the presence of antibodies on the surface of Au-SPEs using multiple techniques, including colorimetry, fluorometry, Ultraviolet-visible (UV-Vis), Fouriertransform infrared (FTIR) spectroscopy, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis [44]. The digital images captured for the unmodified (i.e., no PEG and/or antibody) and PEG/antibody-modified Au-SPEs showed a change in the color of the modified electrode surface (Figure 1b). In addition, the Au-SPEs were digested using tris (2-carboxyethyl) phosphine (TCEP), and the UV-Vis spectrophotometry technique was used to confirm the presence of antibodies in the eluted solution [45][46][47]. The characteristic absorbance peak for PEG was observed near 220 nm, and the absorbance peak for the antibody was observed near 260 nm, compared with unmodified control Au-SPEs (Figure 2c). To further characterize the anti-KPC-2 IgG antibody-modified Au-SPEs, a horseradish peroxidase (HRP)-based colorimetry technique was used. The unmodified and anti-KPC-2 IgG-modified electrodes were incubated with HRP-conjugated anti-IgG for 15 min, and then they were gently washed three times, and then 10 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to the Au-SPEs (Figure 2a). The complex was incubated with TMB for 10 min, and a strong blue color was seen in the antibody-modified SPE. This suggests that the HRP-bound anti-IgG was able to successfully bind the anti-KPC-2 antibody and was able to reduce the TMB to produce a blue color. The TMB added to the unmodified SPE did not produce any color change, indicating the absence of anti-KPC-2 antibodies. Further, the reduced TMB substrate was collected, and the absorbance of the formed complex was measured at 652 nm. The sharp rise in the absorbance at 652 nm confirmed the presence of anti-KPC-2 antibodies on the surface of Au-SPE and its labeling with HRP. The KPC-2 antibody-modified Au-SPEs were also incubated with protein-G coupled with fluorescein isothiocyanate (FITC) for 15 min (Figure 3a). Protein G tends to bind to most IgG molecules at near physiological pH at room temperature (25 °C). The bound antibody complex was then digested using TCEP from both unmodified and modified SPEs to release the IgG-protein G-FITC complex from the surface of Au-SPE, and the fluorescence signal was measured for the released complex at 528 nm. The coupled FITC to protein G was able to produce a fluorescent signal at 528 nm, which was ~68.2% higher than the control sample of unmodified electrodes, indicating a successful conjugation of antibody to the surface of Au-SPE.   To test the efficiency of the antibody-modified SPEs to capture the target KPC-2 enzyme, antibody-modified Au-SPEs were used to capture carbapenemases (at a very low concentration of 20 ng/mL). The SPEs were incubated with the KPC-2 β-lactamase for 30 min and washed 3 times using 10 mM phosphate buffer pH 7.2 to remove excess unbound enzyme. The complex was then digested using TCEP, and the released complex was used to perform SDS-PAGE and FTIR analyses. FTIR analysis results showed absorption bands of the amide I group at 1625 cm −1 (C−O stretching vibration of peptide linkages) and the COOH group (C=O stretching vibration) at around 1784.05 cm −1 (Figure 4a), indicating a successful surface modification with antibody and enzyme capture using the modified Au-SPEs [48][49][50]. The TCEP-digested complex from the electrodes' surface was also analyzed using the SDS-PAGE technique. The presence of a band specific to KPC-2 antigen at 27.2 KDa in the TCEP digested sample indicates successful and effective capture of the KPC-2 β-lactamase by the antibody coated on the surface of Au-SPE. In addition, we used UV-Vis spectroscopy to compare the capture efficiency of KPC-2 enzyme on the surface of Au-SPE modified using the developed PEG protocol with the traditional NHS-based chemistry that relies on using succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The NHS ester reacts with the amine groups of anti-KPC-2 IgG antibodies, and the pyridyldithiol reactive groups in the generated SPDP-modified antibody molecules bind to the surface of Au-SPEs. The results indicated a 12.6 ± 2.1 fold increase in the capture of the target KPC-2 enzyme using our PEG-based site-specific direct conjugation approach (Figure 4c). To test the efficiency of the antibody-modified SPEs to capture the target KPC-2 enzyme, antibody-modified Au-SPEs were used to capture carbapenemases (at a very low concentration of 20 ng/mL). The SPEs were incubated with the KPC-2 β-lactamase for 30 min and washed 3 times using 10 mM phosphate buffer pH 7.2 to remove excess unbound enzyme. The complex was then digested using TCEP, and the released complex was used to perform SDS-PAGE and FTIR analyses. FTIR analysis results showed absorption bands of the amide I group at 1625 cm −1 (C−O stretching vibration of peptide linkages) and the COOH group (C=O stretching vibration) at around 1784.05 cm −1 (Figure 4a), indicating a successful surface modification with antibody and enzyme capture using the modified Au-SPEs [48][49][50]. The TCEP-digested complex from the electrodes' surface was also analyzed using the SDS-PAGE technique. The presence of a band specific to KPC-2 antigen at 27.2 KDa in the TCEP digested sample indicates successful and effective capture of the KPC-2 β-lactamase by the antibody coated on the surface of Au-SPE. In addition, we used UV-Vis spectroscopy to compare the capture efficiency of KPC-2 enzyme on the surface of Au-SPE modified using the developed PEG protocol with the traditional NHS-based chemistry that relies on using succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The NHS ester reacts with the amine groups of anti-KPC-2 IgG antibodies, and the pyridyldithiol reactive groups in the generated SPDP-modified antibody molecules bind to the surface of Au-SPEs. The results indicated a 12.6 ± 2.1 fold increase in the capture of the target KPC-2 enzyme using our PEG-based site-specific direct conjugation approach (Figure 4c).
PEGylation of antibodies affects their affinity for their target receptor. PEG functionalization of antibodies is also known to modify the biological activity of antibodies. Hence, optimal PEGylation (depending on the arm number or arm length of PEG) is necessary as it controls the sensitivity of the antigen-antibody interaction system. For instance, in non-exclusive target recognition by diagnostic probes, it becomes crucial to enhance specific targets to achieve a distinct contrast from the control. In such a case, PEGylation of antibodies has been shown to enhance antigen/ target recognition. A conditional aptamer that was synthesized by conjugating PEG5000-azobenzene-NHS, which was responsive to hypoxia and in turn created a 'caging moiety' causing a conditional recognition of the target [51]. The PEG moiety also endows a 'stealth effect' on the attached antibody, enabling selective targeting of antigen-overexpressing tissues by the antibody [52]. Moreover, the application of the PEG chemistry for detection of cancer-related antigens using screen-printed electrodes was reported much earlier and was a highly sensitive immunosensor with a sensitivity as low as 2.38 pg/mL, thus making the technique of directional conjugation widely accepted [53]. PEGylation of antibodies affects their affinity for their target receptor. PEG functionalization of antibodies is also known to modify the biological activity of antibodies. Hence, optimal PEGylation (depending on the arm number or arm length of PEG) is necessary as it controls the sensitivity of the antigen-antibody interaction system. For instance, in nonexclusive target recognition by diagnostic probes, it becomes crucial to enhance specific targets to achieve a distinct contrast from the control. In such a case, PEGylation of antibodies has been shown to enhance antigen/ target recognition. A conditional aptamer that was synthesized by conjugating PEG5000-azobenzene-NHS, which was responsive to hypoxia and in turn created a 'caging moiety' causing a conditional recognition of the target [51]. The PEG moiety also endows a 'stealth effect' on the attached antibody, enabling selective targeting of antigen-overexpressing tissues by the antibody [52]. Moreover, the application of the PEG chemistry for detection of cancer-related antigens using screenprinted electrodes was reported much earlier and was a highly sensitive immunosensor with a sensitivity as low as 2.38 pg/mL, thus making the technique of directional conjugation widely accepted [53].
In this study, we presented a novel conjugation chemistry for developing enhanced KPC-2 enzyme testing at POC settings using SPEs-based electrochemical immunoassay [54]. Our approach relied on a site-directed reaction that used a heterobifunctional PEG polymer to link oxidized polyclonal anti-KPC-2 antibodies to the surface of Au-SPEs. We achieved PEGylation of the Au-SPE using SH-PEG-hydrazide, which left the hydrazide groups free to react with the oxidized Fc regions of anti-KPC-2 polyclonal antibodies. Our results demonstrated that this site-directed conjugation approach offered a viable alternative to traditional NHS-based chemistry for the modification of Au-SPEs with antibodies. By using directional conjugation, we were able to obtain highly efficient and specific binding of the antibody to the enzyme, which is crucial for designing and developing accurate detection of KPC-2. We also conducted a series of tests to confirm the presence of the an- The surface of Au-SPEs (5 electrodes) was modified with antibody, loaded with 10 µL of KPC-2 enzyme at a concentration of 20 ng/mL, and incubated for 30 min at room temperature. After incubation, the unbound target enzyme was collected, and the concentration of the captured target KPC-2 enzyme was estimated from the total concentration of the loaded KPC-2 enzyme using UV-Vis spectroscopy.
In this study, we presented a novel conjugation chemistry for developing enhanced KPC-2 enzyme testing at POC settings using SPEs-based electrochemical immunoassay [54]. Our approach relied on a site-directed reaction that used a heterobifunctional PEG polymer to link oxidized polyclonal anti-KPC-2 antibodies to the surface of Au-SPEs. We achieved PEGylation of the Au-SPE using SH-PEG-hydrazide, which left the hydrazide groups free to react with the oxidized Fc regions of anti-KPC-2 polyclonal antibodies. Our results demonstrated that this site-directed conjugation approach offered a viable alternative to traditional NHS-based chemistry for the modification of Au-SPEs with antibodies. By using directional conjugation, we were able to obtain highly efficient and specific binding of the antibody to the enzyme, which is crucial for designing and developing accurate detection of KPC-2. We also conducted a series of tests to confirm the presence of the antibody on the surface of the modified Au-SPEs, including colorimetry, fluorometry, UV-Vis and FTIR spectroscopy, and SDS-PAGE analysis. Our findings demonstrated the successful capture of the KPC-2 enzyme using the modified Au-SPEs, with the antibody-modified Au-SPEs capturing the enzyme at a very low concentration of 20 ng/mL. These promising results may have implications for the development of diagnostic tools for the early detection of infections caused by KPC-2 or other carbapenemase-producing bacteria, which is crucial for the effective management and control of antibiotic-resistant infections. Overall, our study highlights the potential of our PEG-based site-directed conjugation approach for the development of sensitive and specific POC diagnostics for a range of diseases and conditions.